1. Field of the Invention
The present invention relates to integral elements for the essentially dry analysis of total cholesterol in aqueous solutions, such as blood serum.
2. Description of Related Art
Known quantitative analyses of total cholesterol (i.e., the sum of both free and esterified cholesterol) aqueous solutions such as blood serum, have generally involved the handling of corrosive chemicals to hydrolyze the cholesterol esters to free cholesterol (i.e., chemically unreacted or combined cholesterol in its molecular form) and analyze for free cholesterol such techniques are generally complex and not easily automated. In the best known conventional technique, blood serum is extracted with an organic solvent, cholesterol esters in the extract are saponified with alcoholic KOH and free cholesterol is isolated and assayed using known techniques which generally involve the handling of corrosive chemicals such as fuerric perchlorate and sulfuric acid.
The incorporation of reagent sequences of this type into "dry" analytical systems is, quite obviously, very difficult if not impossible.
Belgian Pat. No. 801,742 describes unique integral elements for use in the qualitative and quantitative analysis of liquids such as blood serum and urine, which elements preferably comprise a porous spreading layer in fluid contact or communication with a reagent layer which comprises at least one material interactive with a component or decomposition product of a component of the liquid. This patent includes no suggestion that any total cholesterol assay composition, much less one of the type described herein, could be incorporated into a dry analytical element.
Research Disclosure, Vol. 127, pp. 54-56 (1974), describes a totally enzymatic method for the hydrolysis of cholesterol esters in solution using a lipase having cholesterol esterase activity and a protease. There is no suggestion that the hydrolysis technique can be incorporated into an essentially dry web-form analytical element useful for total cholesterol assay.
Research Disclosure, Vol. 127, pp. 39-42 (1974), describes a totally enzymatic, quantitative, single solution assay for cholesterol in aqueous solutions containing both free and esterified cholesterol using the foregoing cholesterol hydrolysis technique combined with a cholesterol oxidase degradation of free cholesterol. The assay solution may also include a hydrogen peroxide detection system based on a peroxidase containing indicator system of the type which has been used for glucose and uric acid assay. There is no indication in that publication that the assay composition can be used in an essentially dry web-form element.
The preparation of cholesterol oxidase is described in Research Disclosure, Vol. 126, pp. 46-50, (1974), and German Offenlegungsschrift 2,246,695 published Mar. 26, 1973.
German Offenlegungsschrift No. 2,246,695 published Mar. 26, 1973 describes the use of a cholesterol oxidase enzyme different from that described in the aforementioned Research Disclosure, Vol. 126, pp. 46 -50 (1974), to assay for free cholesterol. The method of this German publication is a solution method and still requires the handling of corrosive materials to hydrolyze the cholesterol esters which may be present in blood serum in addition to the sometimes unwieldy handling of solutions to perform the assay. There is no suggestion in either of these references to use cholesterol oxidase in an essentially dry analytical web.
U.S. Pat. No. 3,607,093 to Stone issued Sept. 21, 1971 suggests incorporating cholesterol oxidase into liquid permeable membranes of uniform composition useful in the assay of, for example, biological fluids. There is no suggestion that an assay composition for total cholesterol including free and esterified cholesterol can be incorporated into a membrane of this or any other type.
Belgian Pat. No. 811,728 describes a composition and method for the determination of total cholesterol. The composition comprises a chemical system having cholesterol oxidase activity and a chemical system having cholesterol ester hydrolase activity obtained through the extraction of animal or human pancreas, liver or intestines and means for determining hydrogen peroxide released by the action of the cholesterol oxidase on cholesterol released by the cholesterol ester hydrolase. According to this patent, the cholesterol ester hydrolase requires the presence of a biliary cofactor and specifically prescribes that the test composition be free of proteolytic activity. There therefore exists a need for further simplified assays for total cholesterol, to avoid the undesirable reagent handling and complex chemical compositions and preparations currently required for this assay.